Carotenoids are natural pigments that are useful as feed additives, food additives, cosmetic ingredients, pharmaceutical agents and the like. Examples of carotenoids include astaxanthin, canthaxanthin, zeaxanthin, β-cryptoxanthin, lycopene, β-carotene, phoenicoxanthin, adonixanthin, echinenone, asteroidenone and 3-hydroxyechinenone. Among them, astaxanthin is useful as feed additives, for example, as body color-improving agents for cultivated fishes such as salmon, trout and sea bream or as egg yolk color-improving agents for poultry. Moreover astaxanthin is highly valuable in the industries as safe natural food additives, healthy food materials, and cosmetic ingredients. Similar to astaxanthin, adonixanthin and phoenicoxanthin are also expected of their use as feed additives, food additives, cosmetic ingredients, pharmaceutical agents and the like once their industrial production methods are established. Furthermore, β-carotene is used as feed additives, food additives, cosmetic ingredients, pharmaceutical agents and the like; canthaxanthin is used as feed additives, food additives, cosmetics and the like; and zeaxanthin is used as food additives, feed additives, cosmetic ingredients and the like. In addition, lycopene, echinenone, β-cryptoxanthin, 3-hydroxyechinenone and asteroidenone are also expected of their use as feed additives, food materials, cosmetic ingredients and the like. Methods for producing these carotenoids include chemical synthesis processes, extraction from natural sources, production methods using microorganisms and the like.
As methods for chemically synthesizing astaxanthin, a method utilizing conversion of β-carotene (Non-patent Document 1: Pure Appl. Chem, 57, 741, 1985) and a method employing synthesis from C15 phosphonium salt (Non-patent Document 2: Helv. Chim. Acta, 64, 2436, 1981) are known. In addition, since astaxathin is present in fishes such as sea bream and salmon as well as crustaceans such as shrimp, crab and krill, it may also be extracted therefrom. Chemically synthesized astaxanthin is commercially available as a feed additive.
Methods that have been reported for producing astaxanthin using microorganisms include cultivation with green alga Haemaococcus pluvialis (Patent Document 1: Japanese Laid-Open Patent Publication No. 2007-97584), a fermentation process using red yeast Phaffia rhodozyma (Patent Document 2: Japanese Laid-Open Patent Publication No. H 11-69969), a fermentation process using a bacterium belonging to genus Sphingomonas (Patent Document 3: Japanese Laid-Open Patent Publication No. 2006-191919), a fermentation process using a bacterium belonging to genus Brevundimonas (Patent Document 4: Japanese Laid-Open Patent Publication No. 2006-340676), a fermentation process using a bacterium belonging to genus Erythrobacter (Patent Document 5: Japanese Laid-Open Patent Publication No. 2008-259452), and a fermentation process using a bacterium belonging to genus Paracoccus (hereinafter, also referred to as a “Paracoccus bacterium”). Examples of bacteria belonging to astaxanthin-producing genus Paracoccus include strains E-396 and A-581-1 (Patent Document 6: Japanese Laid-Open Patent Publication No. H 7-79796 and Non-patent Document 3: International Journal of Systematic Bacteriology (1999), 49, 277-282). Examples of other bacteria belonging to astaxanthin-producing genus Paracoccus include Paracoccus marcusii strain MH1 (Patent Document 7: Publication of Japanese translation of PCT international application No. 2001-512030), Paracoccus haeundaensis strain BC74171 (Non-patent Document 4: International Journal of Systematic and Evolutionary Microbiology (2004), 54, 1699-1702), Paracoccus bacterial strain N-81106 (Patent Document 8: Japanese Laid-Open Patent Publication No, 2007-244205), Paracoccus zeaxanthinifaciens (Non-patent Document 5: International Journal of Systematic and Evolutionary Microbiology (2003), 53, 231-238) and Paracoccus sp. strain PC-1 (Patent Document 9: WO 2005/118812).
There have been, however, several problems concerning the above-mentioned carotenoid production methods. For example, chemical synthetic methods have unfavorable impression on consumers from a safety perspective. Extraction from natural sources is associated with high production costs. In addition, production using a green alga or yeast is low in productivity and has difficulty in extraction of a carotenoid therefrom due to strong cell walls thereof.
Meanwhile, bacteria belonging to genus Paracoccus are advantageous in that their proliferation rates are rapid, their carotenoid productivity is high, and carotenoids can readily be extracted therefrom, etc. However, Paracoccus bacteria partially secrete carotenoids as vesicles outside the cells, and thus many carotenoids are dispersed as microparticles in the liquid. Therefore, it is difficult to remove supernatant from the culture and collect carotenoids at high yield. Conventional well-known carotenoid-producing bacteria beside Paracoccus bacteria, namely green algae Haematococcus pluvialis and red yeast Phaffia rhodozyma, have no such problem since carotenoids are entirely accumulated in the cells. In a culture of a carotenoid-producing Paracoccus bacterium, extracellular carotenoids may amount to as much as 80% of the entire carotenoids including the intracellular carotenoids. A method has been proposed for separating carotenoids from a culture of Paracoccus bacterium, which includes: centrifuging the culture at a first rate that is sufficient to pelletize the cells; collecting the supernatant containing carotenoids; and centrifuging the supernatant at a second rate that is sufficient to pelletize the carotenoid vesicles (Patent Document 4: Publication of Japanese translation of PCT international application No. 2001-512030). However, since carotenoids are present as very small particles in the culture supernatant, pelletization requires ultrafast centrifugation at a level of 100,000×g, which is impractical considering the costs of equipment and power.